Genetic and biochemical characterization of waxy mutants in cereals.

During fine structure analyses of artificially induced waxy mutants in maize by use of Nelson's pollen analysis methods, it was noticed that some mutants showed a phenotype intermediate between waxy and normal nonwaxy. Such intermediate or leaky waxy mutants were frequent among mutants induced by a chemical mutagen, EMS. They seemed to be located evenly within the waxy locus. This would suggest that they might have been missense mutations, which would produce full-sized but partially inactivated enzymes, rather than deletions or frameshift mutations. To confirm the intermediate phenotype, a rapid measuring system was developed to measure waxiness of endosperm quantitatively. It was based on the blue value method and is applicable to a single grain of rice. Among the 27 waxy mutant lines of maize, including 11 EMS-induced, two of the EMS-induced mutants were clearly intermediate. Eighteen EMS induced wx mutants of rice were also examined, and nine were intermediate. ImagesFIGURE 1.


Introduction
Pollens of higher plants have many advantages in genetical analyses. They are haploid germ cells and can be examined in very large population if appropriate genetic trait is used. Fine structure analyses of waxy (wx)(1-3) and amylose extender (ae) (4) in maize as well as glutinous (gl: comparable to wx) in rice (5) are examples of successful analyses utilizing the merits of pollen analysis. Intralocus mapping of mutant alleles would be a good means to see possibilities of the presence of point mutations in higher plants. To obtain materials for such analyses, many wx mutants have been induced in maize by using ionizing radiations, germicidal ultraviolet light, or chemical mutagens (6)(7)(8). In these experiments, wx mutants were detected after a test cross of the treated materials with a standard tester wx stock. In the case of ultraviolet light, mutants were detected after self-pollination, but later they were crossed to the tester wx to confirm allelism. Therefore all of these waxy mutants belonged to the wx *Department of Induced Mutation, National Institute of Genetics, Misima, Sizuoka-ken, Japan. January 1981 locus on chromosome 9. During fine structure analyses of these wx mutants by using Nelson's pollen analysis method (2), it was noticed that some mutants showed intermediate phenotype between wx and normal nonwaxy (Wx) (9). An example of such an intermediate wx mutant is shown in Figure 1. Here the intermediate wx mutant had been crossed to one of the standard wx mutant (wx90), and segregating pollen were stained with iodine. The dark pollen at the center is considered to be a nonwaxy recombinant which may serve as Wx color standard in the same preparation. Other pollen grains segregated into light-colored wx and intermediate-colored mutant pollen in a theoretical one to one ratio. Those intermediate wx mutants were stable in their degree of waxyness. Such intermediate or leaky waxy phenotypes were inherent in some of the mutants induced by a chemical mutagen, ethyl methanesulfonate (EMS). They seemed to be located evenly within the waxy locus (9). They might have been missense mutations which might produce protein or enzyme with normal length but its function partially inactivated. To confirm intermediate phenotypes, a rapid measuring system was developed to measure waxyness of _ *s~~~. -1 !^ws. W.. endosperm starch quantitatively. It was based oil the blue value method to measure amylose fraction of starch (10). Results of measurements of wvx mutant lines in maize and also in rice are reported here. Distribution characteristics of the waxyness indices were also discussed.

Plant Materials
Normal nonwaxy (Wx) strain of maize (6311R) used was a multiple dominant genetic stock with purple plant color. Standard waxy (wx) mutant strain of maize (639) was a multiple recessive genetic stock with C sh1 and bz marker genes. These stocks were originally obtained from Dr. H. H. Smith, Brookhaven National Laboratory. Other genetic stocks were kindly supplied by Dr. R. W. Briggs (Funk Seeds International), Dr. 0. E. Nelson (University of Wisconsin), and Maize Genetics Cooperative. Other waxy mutants were induced in the strain 6311R by seed treatment with radiations or EMS, or pollen irradiation by germicidal ultraviolet light. Most of the materials were used with 6311R background.
The standard waxy starch mutant of rice, 504T65wx, was obtained from Dr. H. Morishima (National Institute of Genetics). Other wx mutants in rice examined were induced by EMS treatment of seeds of a normal nonwaxy Japonica variety, Norin No. 8 (11, 12). These materials were selfpollinated and selected for normal growth for a few generations. 36

Preparation of Starch Solution
Measurements were done on a kernel basis. Each whole maize kernel was crushed to coarse meal. The kernel was placed in a plastic cylinder on a piece of iron plate. A short iron rod was inserted into the cylinder and was hit with a hammer. The meal was transferred to a test tube. A 5-ml portion of distilled water was added to each tube. For rice, hulls were removed, and each grain was crushed with pliers and put into a test tube. A 3-ml portion of water per tube was used for rice. The test tubes were autoclaved at 1 atm for 15 min. After stirring for several seconds while hot, they were left overnight for cooling and sedimentation of debris. The supernatant fluid was used in the measurement.

Iodine Reagent and Staining
Half strength of Nelson's 12-KI solution (2) was used to stain the starch solution. The recipe adopted was 450 mg iodine and 2.5 g KI in 500 ml of water. To reduce the effect of color of excess iodine reagent, staining was done in excess of starch. A 1-ml portion of sample starch solution was mixed with 0.1 ml of iodine reagent and 2 ml of water in a 10 mm x 10 mm glass absorption cell. The cell was immediately set in the colorimeter. The measurement was completed within 1 min.

Colorimetry
The colorimetric apparatus was designed to measure with two wavelengths (430 nm and 660 nm) simultaneously (13,14). As shown in Figure 2, it FIGURE 2. Schematic of colorimeter. Absorption cell is equipped with inlet, outlet tubes and a stirring blade for dilution. Light beam was divided by mirrors and measured by silicon photocells behind interference filters.
Environmental Health Perspectives consisted of an incandescent lamp, an absorption cell, mirrors to divide the light beams, interference filters, and silicon photocells of photometry grade (Hamamatsu TV, S780-8BK). A small mixing blade and inlet and outlet capillaries were inserted into absorption cell so that continuous dilution could be done while concentration was monitored photometrically. When the sample solution was diluted to 50% transmittance for 430 nm, an electric signal was produced to command a digital printer to print out the transmittance value for 660 nm. This transmittance value for 660 nm was used as index to express waxy phenotype. Monochromatic lights obtained by using interference filters were used for high reproducibility.
ranged from 63 to 70. In rice, the values were a little higher both in Wx and wx lines. Figures 3 and  5 show distribution of wx indices of each kernel of the wx mutants of maize and rice respectively. High reproducibility of the measurements is demonstrated in strain 6311R (Wx) in Figure 3. M14, another Wx inbred line, showed wider distribution of the waxy indices probably inherent to this line. In Figures 4 and 6, wx mutants were placed in orders of mean wx indices of each wx mutant line.
In maize, most of the 27 wx mutant lines examined were complete wx mutants (Fig. 4). Eight wx mutant lines were genetic stocks of translocation or other phenotypes. Those wx genes might be presumably of the same origin. 1J2, R, A39, 1M2, 90

Results
The rationale of the photometry at two wavelengths is to measure amylose which is deleted in the wx phenotype, by the blue value method at a certain concentration of starch solution which was determined photometrically. Amylopectin is a major and common fraction in both types of starches. In Wx starch, amylopectin amounted to about 75% and in wx starch it reached almost 100% (15)(16)(17). In solution, amylopectin is stained reddish purple by iodine (18). To bring the concentration of sample solution to a predetermined level, the iodinestained solution was diluted continuously while monitoring the concentration of iodine-amylopectin complex by 430 nm blue light. When the concentration reached to 50% transmission at 430 nm, the percent transmission (%T) value at 660 nm red light was measured and recorded by a digital printer. This %T value at 660 nm represents the concentration of the blue colored amylose-iodine complex.
For preparation of the starch solution, alkaline dispersion was also tested. Starch could be dispersed well from the clushed endosperm by 1N KOH solution. Howe ver, it was not used in the present measurements for two reasons: first, it requires neutralization of each sample solution before iodine staining; second, it reduced the difference of indices between Wx and wx. Compared to alkaline dispersion, autoclaving was convenient and good for uniform preparation of many sample solutions. The time of autoclaving was varied, and no significant difference was found from 5 to 30 min. A period of 15 min was adopted because it is the same condition as routine sterilization procedure in our laboratory. Measurements of waxy phenotype were made for each kernel.  and H21 are known to be mutants of independent origins. 75UVwxl and 75UVwx2 were mutants induced by irradiation of pollen grains with germicidal ultraviolet light. Mutants with prefixes 71. 72, and 74 were EMS-induced mutants. Of 11 EMSinduced wx mutants, two were clearly intermediate (Fig. 3) and another two were next to them (Fig. 4). In rice, some wx mutants were very close to normal Wx (Figs. 5 and 6). The mutant 74wx3 had been found as doubtful wx mutants. Examination of waxy indices on a kernel basis revealed that the distribution of the indices was extended to higher region compared to normal Wx, indicating that amylose content was affected to some extent, though the mean value of the indices did not differ significantly. Here, the line 74wx3 was considered very leaky wx mutant. Among the 18 EMS-induced wx mutant lines, nine could be classified as intermediate wx mutants.
Compared to maize, in which wx mutants were detected after a test cross with a standard wx line, wx mutant in rice were detected in M2 endosperms 38

Discussion
The nature of mutation in higher plants has been studied extensively by many investigators in related fields. After the suggestion by Stadler and Roman (19) 8  74wx3  75wx3  74wx5  74wx8  76wx3  75wx2  74wx2  75wxl  74wx7  76wx2   74wxl  75wx5  504 T 65 wx  76wx5  74wx9  wx73-1  76wxl  74wx6  However, induction of a point mutation in its strict definition, singje base-pair substitution, and its confirmation became possible by the recent developments of studies on chemical mutagenesis (20)(21)(22). Detailed analyses of mutant characters and knowledge of biochemistry concerning both in chemical reactions in mutagenesis (23)(24)(25) and gene function (26) would support this view. Intralocus fine structure analysis developed by Nelson (1) would have limited resolution in measuring the size of genetic alteration which caused the mutation. Immunological detection of CRM and electrophoretic detection of modified protein might be a good means to show that the mutation concerned was a missense type base pair substitution. These methods were successfully applied to sh1 locus mutants in maize (27).
CRM and modified proteins were detected in some EMS-induced sh1 mutants. Intermediate or leaky phenotypes reported here would be another examples to indicate, though indirectly, the presence of full size protein molecules which is affected its function only partially. The intermediate phenotypes reported here in maize were seemed to be inherent to certain EMS induced wx mutant lines. These wx mutant genes were allelic to other wx mutants suggesting that they were in the same wx cistron.
A modifier of Wx-wx phenotype, amylose extender (ae), is known on a different chromosome. It is a recessive gene and increases the amylose or amyloselike fraction in starch both in Wx or wx January 1981  Distribution of induced wx mutants as placed by Wx pollen grain frequencies in hybrids between tester wx and the mutants. Arbitrary grading of iodine stain was made between (1) no background segregation, and (4) half of the wx pollen stained so dark that scoring of Wx needed extra care in staining [from Amano (9)].
phenotypes (15,16,28). But the present examples of the intermediate wx mutant line are not of this case. There was no phenotypic expression of ae-wx double mutations (15), and no ae-Ae segregation was observed. It can be also noticed from Figure 7 that there are too many intermediate wx mutants induced by EMS to permit them to be ascribed to simultaneous mutation at ae locus. In Figure 7, besides the recombinational distances from the standard wx stock, 639, expressions of wx phenotype as observed after iodine staining of pollen grains were classified into four groups. The 39 * X * -classifications were made between colors of standard wx and normal Wx recombinant pollen grains in the same slide preparation.  Figure 6. Mean wx indices for each wx mutant line were distributed rather continuously from the Wx region (74wx3)to fully wx region (74wx7). Since in these analyses all viable wx mutants were analyzed, it might be comparable to the results shown in Figure 7, as for sampling of materials. In both materials, EMS treatments were made under conditions favorable for less chromosome aberration (29). Simply, EMS was dissolved in distilled, deionized water without use of any buffering salts. The solution was acidic and the pH was around 3. High efficiency in induction of mutation and good survival of the induced mutant (7,9) would support the explanation of intermediate wx phenotype by missense mutation. Intermediate phenotypes were also observed in some of the non-CI (9) or sh1 mutations. These facts would suggest that an intermediate phenotype might be a rather common phenomenon where point mutations could occur.
In conclusion, it is advised that the monitoring system for mutagenic activity of environmental pollutants be prepared also for intermediate mutant phenotypes if point mutation inducers are expected.